Lung cancer is the leading cause of cancer death in the United States. Adenocarcinoma, the histological subtype most frequently seen in never smokers and former smokers, is now the most common type of lung cancer in men and women. The increasing incidence of lung adenocarcinoma underlines the importance of understanding the development and progression of this lethal disease. DMA hypermethylation at promoter CpG islands is a key mechanism for tumor suppressor gene inactivation in cancer. Using a unique collection of pre-invasive lesions, lung adenocarcinomas of clinical stages IA-IIIA, and control lung samples, we propose to undertake a genome-wide search for DNA methylation changes associated with adenocarcinoma development and progression. The Specific Aims of this study are: 1) To identify new epigenetic changes in lung adenocarcinoma compared to histologically normal lung by genome-wide DNA methylation profiling. We will compare early and late stage adenocarcinoma and non-cancer lung to identify concurrent changes in methylation and expression, using CpG island and expression microarrays. Select loci will be verified by bisulfite sequencing and quantitative RT-PCR. 2) To determine whether DNA methylation changes for loci identified in Aim 1 occur during development and progression of lung adenocarcinoma. Using the high throughput system MethyLight, we will compare methylation profiles in 50 archival samples each of stages IA, IB, IIA, IIB and MIA lung adenocarcinoma, in pre-invasive lesions, and in non-tumor lung. 3) To determine whether correlations exist between the methylation profiles observed in Aim 2 and patient survival. 4) To examine the biological significance of loci identfied under Aim 2 using functional experiments in vitro: forcing expression of genes or artifically silencing them by RNAi. The effects of these manipulations on cell lines representing precancerous and cancerous lesions will be evaluated by measuring proliferation rates, cell cycle distribution, cell mobility, anchorage-independent growth and the ability to form tumors in nude mice. Over 150,000 Americans die every year from lung cancer. Early detection is the best way to prevent cancer deaths. The identification of sequential epigenetic alterations that occur during lung adenocarcinoma development could provide new markers for early detection and prognostication as well as possible new targets for drug development, thereby saving or extending the lives of thousands of lung cancer patients.